+

WO1997019178A2 - Proteases metalliques autoantigenes et procedes permettant de diagnostiquer des maladies auto-immunes - Google Patents

Proteases metalliques autoantigenes et procedes permettant de diagnostiquer des maladies auto-immunes Download PDF

Info

Publication number
WO1997019178A2
WO1997019178A2 PCT/DE1996/002094 DE9602094W WO9719178A2 WO 1997019178 A2 WO1997019178 A2 WO 1997019178A2 DE 9602094 W DE9602094 W DE 9602094W WO 9719178 A2 WO9719178 A2 WO 9719178A2
Authority
WO
WIPO (PCT)
Prior art keywords
polypeptide
nucleic acid
autoantigenic
acid sequence
autoimmune diseases
Prior art date
Application number
PCT/DE1996/002094
Other languages
German (de)
English (en)
Other versions
WO1997019178A3 (fr
Inventor
Ulrich Krawinkel
Simon Mauch
Radislav Sedlacek
Original Assignee
Universität Konstanz
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Universität Konstanz filed Critical Universität Konstanz
Publication of WO1997019178A2 publication Critical patent/WO1997019178A2/fr
Publication of WO1997019178A3 publication Critical patent/WO1997019178A3/fr

Links

Classifications

    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/48—Hydrolases (3) acting on peptide bonds (3.4)
    • C12N9/50—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25)
    • C12N9/64—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from animal tissue
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/564—Immunoassay; Biospecific binding assay; Materials therefor for pre-existing immune complex or autoimmune disease, i.e. systemic lupus erythematosus, rheumatoid arthritis, multiple sclerosis, rheumatoid factors or complement components C1-C9
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • C07K14/4701—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals not used
    • C07K14/4713—Autoimmune diseases, e.g. Insulin-dependent diabetes mellitus, multiple sclerosis, rheumathoid arthritis, systemic lupus erythematosus; Autoantigens

Definitions

  • the present invention relates to nucleic acids which contain at least one nucleic acid sequence coding for a polypeptide, the polypeptide containing at least one autoantigenic determinant originating from a metal protease, the polypeptide itself and antibodies directed against the polypeptide and specific directed against the polypeptide Inhibitors. Furthermore, the present invention relates to methods and kits for the detection of autoantigenic metal proteases and autoimmune diseases such as rheumatoid arthritis
  • the present invention is based on the object ⁇ determine a new system for diagnosing autoimmune diseases, which overcomes the disadvantages of the examination methods presently used in the prior art and insbe ⁇ sondere overall eicrnet for screening for autoimmune diseases
  • This object is achieved by the embodiments of the present invention which are characterized in the claims .
  • a first subject of the invention relates to a nucleic acid which contains at least one nucleic acid sequence coding for a polypeptide, the polypeptide comprising at least one autoantigenic determinant derived from a metal protease.
  • nucleic acid and nucleic acid sequence mean natural or semisynthetic or synthetic or modified nucleic acid molecules made from deoxyribonucleotides and / or ribonucleotides and / or modified nucleotides.
  • polypeptide encompasses naturally occurring polypeptides and recombinant polypeptides.
  • Naturally occurring polypeptides according to the invention comprise autoantigenic metal proteases per se or autoantigenic sections or fragments thereof.
  • the metal proteases are so-called membrane proteins, i.e. they adhere to the cell surface membrane in such a way that the functional parts of the protein face outwards and can optionally be cleaved off proteolytically.
  • Metal proteases preferably originate from macrophages and related cell types and occur, for example, in the inner skin of the joint, Afembrana synovialis, from mammals.
  • Recombinant polypeptides refer to a construct made using molecular biological techniques, which is based on the natural DNA of the original genome or the natural DNA modified with a foreign DNA sequence and can be recombined, e.g. with plasmids, and can be replicated and expressed in a suitable host system.
  • autoantigenic determinant or epitope means that one or more linear or conformational regions of the Metal proteases are recognized by one's own immune system by so-called autoantibodies.
  • autoantibodies The presence of autoantibodies is regarded as an indication that a protein of one's own cells, for example in the macrophages of the synovial membrane, is expressed quantitatively and qualitatively abnormally as a result of a disease.
  • the possible cause of joint destruction in rheumatoid arthritis is assumed to be that an as yet unidentified infectious agent stimulates joint macrophages for the increased expression of proteases which release further potential autoantigens through proteolysis of joint tissue (cf. Opdenakker et al., 1994, Immunol.
  • the nucleic acid comprises the nucleic acid sequence shown in FIG. 1 or sections or fragments thereof. In a particularly preferred embodiment of the present invention, the nucleic acid comprises the sequence section 1110-2206 of the nucleic acid sequence shown in FIG. 1.
  • Another object of the present invention is a vector which contains the nucleic acid according to the invention defined above for the expression of the recombinant polypeptide in prokaryotic or eukaryotic host cells.
  • the vector according to the invention can preferably contain suitable regulatory elements, such as promoters, enhancers, termination sequences.
  • the vector according to the invention can for example be an expression vector or a vector for preferably stable integration of the nucleic acid according to the invention into the genetic material of a host cell.
  • a suitable Expressi ⁇ onssystem includes, for example, the vector pET-24 and E. coli BRMDE3) or BL21 (DE3), or for expression in eukaryonti ⁇ 's cells the vector pcDNA3 and cos cells or CHO cells.
  • Another object of the present invention is a host cell which contains the nucleic acid according to the invention or the vector according to the invention.
  • Suitable host cells are for example prokaryotes such as E. coli or eukaryotic host cells such as cos or CHO. In principle, any commercially available pro- or eukaryotic expression system can be used.
  • the nucleic acid according to the invention is either stably integrated in the genetic material of the host cell or the vector according to the invention contains suitable regulatory areas for replication, transcription and / or translation in vivo and / or in vitro, ie also in the cell-free system.
  • polypeptide itself, which is encoded by the nucleic acid sequence defined above, it being possible for the nucleic acid sequence to be degenerate in accordance with the genetic code.
  • the polypeptide according to the invention can be modified in vitro by post-translational reactions in vivo or by chemical and / or enzymatic methods known in the prior art.
  • the polypeptide comprises the amino acid sequence shown in FIG. 2 or sections or fragments thereof, for example the expression product encoded by sequence section 1110-2206 of the nucleic acid sequence shown in FIG.
  • nucleic acid sequence according to the invention, the vector according to the invention and the polypeptide according to the invention can be produced by methods known in the prior art (cf. Ausubel et al., 1991, Current Protocols in Molecular Biology, Wiley Interscience, New York, N.Y.).
  • Another object of the present invention is a po- lyklonaler, preferably monospecific, or monoclonal antibody against the above-defined polypeptide ge ⁇ is directed.
  • the antibody of the invention can be produced by methods known in the art (see Coligan et al., 1993, Current Protocols in Immunology, Wiley Interscience, New York, NY).
  • the present invention further relates to a method for the detection of autoimmune diseases, in particular rheumatoid arthritis (RA) and other autoimmune forms of arthritis such as SLE (systemic lupus erythematosus), MCTD (mixed collagenosis) and PSS (progressive scleroderma), in which either the above defined polypeptide or the above defined antibody with serum from a mammal are subjected to an enzyme immunoassay, preferably an ELISA.
  • RA rheumatoid arthritis
  • SLE systemic lupus erythematosus
  • MCTD mixed collagenosis
  • PSS progressive scleroderma
  • the detection of autoantibodies against metal proteases in the ELISA has the advantages of being non-invasive and considerably less expensive than other diagnostic methods for rheumatoid arthritis, such as X-ray examination or joint puncture.
  • autoantibodies can already be detectable if imaging methods do not yet indicate damage to the joint.
  • specific inhibitors of the metal protease can be used in pharmaceutical formulations for therapy in the joint of a rheumatism patient.
  • Another object of the present invention is a diagnostic kit for the detection of an autoimmune disease, which contains at least either the above-defined polypeptide or the above-defined antibody.
  • FIG. 2 (A) is the 508 AS long primary sequence of the metal protease MPRS, which is encoded by the DNA sequence shown in FIG. 1 (molecular weight, calculated from the 57.4 kDa cDNA sequence).
  • FIG. 2 (B) shows structural properties of this metal protease.
  • FIG. 3 is a Coomassie blue-colored SDS polyacrylamide gel with recombinant MPRS isolated from mononuclear blood cells and its RASED fragment, which comes from an RA patient
  • FIG. 5 shows a homology comparison of the metal protease according to the invention shown in FIG. 2 with previously known metal proteases.
  • Bold amino acids mark strongly conserved areas.
  • the domains typical for metal proteases hydrophobic signal peptide, cleavable propeptide, catalytic domain with Zn 2+ site, "Hmge” region and Hemopexm-like domain
  • MMP-1 membrane type Ma ⁇ trix metalloprotemase
  • the isolation of mRNA from synovial membrane cells of an RA patient is carried out according to methods known in the prior art, and an expression cDNA library is produced using the lambda Zap II expression vector (from Stratagene).
  • the screening (screening) of the cDNA library is carried out with purified immunoglobulin G from the RA patient.
  • a primary clone "Rased” which comprises a fragment of 1106 bp of the metal protease (MPRS) mRNA, was obtained from the cDNA library (3 ⁇ 10 5 clones / ⁇ g mRNA), this fragment contains an autoantigenic epitope based on the screening with immunoglobulin G of the RA patient.
  • MPRS metal protease
  • MPRS mRNA is detected in peripheral mononuclear blood cells via reverse transcription / polymerase chain reaction.
  • the isolation of the mRNA from peripheral mononuclear blood cells and the production of a cDNA bank with lambda Zap II is carried out as described above.
  • the cDNA library is screened with a labeled DNA probe based on the RASED clone.
  • the database comparison shown in FIG. 5 shows at the protein level between 30 and 35% identity (with a similarity of over 50%) to known metal proteases, the catalytic domain and the domain structure typical of metal proteases being present.
  • the expression and purification of MPRS from E. coli is carried out with pET24 and with N-terminal oligo-histidine "tag".
  • the expression and purification of RASED from E. coli is performed with pGEX 1 as a fusion protein with glutamine-S-transferase.
  • the peptide constructs thus obtained were electrophoretically separated using an SDS polyacrylamide gel and the separated peptide constructs were detected by Coomas blue staining of the SDS polyacrylamide gel; see. Figure 3 '.-

Landscapes

  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Organic Chemistry (AREA)
  • Biomedical Technology (AREA)
  • Molecular Biology (AREA)
  • Immunology (AREA)
  • Genetics & Genomics (AREA)
  • Zoology (AREA)
  • Biochemistry (AREA)
  • Wood Science & Technology (AREA)
  • Medicinal Chemistry (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • General Health & Medical Sciences (AREA)
  • Hematology (AREA)
  • Microbiology (AREA)
  • Biotechnology (AREA)
  • Rheumatology (AREA)
  • Urology & Nephrology (AREA)
  • General Engineering & Computer Science (AREA)
  • Rehabilitation Therapy (AREA)
  • Analytical Chemistry (AREA)
  • General Physics & Mathematics (AREA)
  • Diabetes (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Biophysics (AREA)
  • Pathology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Toxicology (AREA)
  • Physics & Mathematics (AREA)
  • Food Science & Technology (AREA)
  • Cell Biology (AREA)
  • Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
  • Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
  • Peptides Or Proteins (AREA)
  • Enzymes And Modification Thereof (AREA)

Abstract

L'invention concerne des acides nucléiques, qui contiennent au moins une séquence d'acide nucléique codant un polypeptide. Le polypeptide contient au moins un déterminant autoantigénique issu d'une protéase métallique. L'invention concerne également ledit polypeptide, des anticorps dirigés contre le polypeptide et des inhibiteurs spécifiques dirigés contre le polypeptide. L'invention concerne en outre des procédés et des nécessaires permettant de détecter la présence de protéases métalliques autoantigéniques et de maladies auto-immunes telles que l'arthrite rhumatoïde.
PCT/DE1996/002094 1995-11-20 1996-11-04 Proteases metalliques autoantigenes et procedes permettant de diagnostiquer des maladies auto-immunes WO1997019178A2 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
DE19543265A DE19543265A1 (de) 1995-11-20 1995-11-20 Autoantigene und Verfahren zur Diagnose von Autoimmunkrankheiten
DE19543265.7 1995-11-20

Publications (2)

Publication Number Publication Date
WO1997019178A2 true WO1997019178A2 (fr) 1997-05-29
WO1997019178A3 WO1997019178A3 (fr) 1997-09-25

Family

ID=7777951

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/DE1996/002094 WO1997019178A2 (fr) 1995-11-20 1996-11-04 Proteases metalliques autoantigenes et procedes permettant de diagnostiquer des maladies auto-immunes

Country Status (2)

Country Link
DE (1) DE19543265A1 (fr)
WO (1) WO1997019178A2 (fr)

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1997040157A1 (fr) * 1996-04-25 1997-10-30 Takeda Chemical Industries, Ltd. Metalloprotease matricielle
WO1998040475A1 (fr) * 1997-03-11 1998-09-17 Abbott Laboratories Gene humain de metalloprotease matricielle, proteines codees a partir de ce dernier, et procedes d'utilisation associes

Family Cites Families (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JPH085920B2 (ja) * 1991-01-21 1996-01-24 富士薬品工業株式会社 抗ヒトストロムライシンモノクローナル抗体及び酵素免疫測定法
DE69333167T2 (de) * 1992-10-29 2004-06-09 Bayer Corp. Diagnostischer test spezifisch für die latente matrix metalloproteinase no. 9
EP0685557B1 (fr) * 1993-11-30 1998-08-12 Fuji Yakuhin Kogyo Kabushiki Kaisha Nouvelle metalloprotease et adn codant pour cette derniere
EP0750672B1 (fr) * 1994-03-17 2001-11-28 Max-Delbrück-Centrum Für Molekulare Medizin Sequences d'adn pour metalloproteases matricielles, leur production et leur utilisation

Cited By (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1997040157A1 (fr) * 1996-04-25 1997-10-30 Takeda Chemical Industries, Ltd. Metalloprotease matricielle
US6566116B1 (en) 1996-04-25 2003-05-20 Takeda Chemical Industries, Ltd. Matrix metalloprotease
US7001997B2 (en) * 1996-04-25 2006-02-21 Takeda Chemical Industries, Ltd. Proteins, their production and use
WO1998040475A1 (fr) * 1997-03-11 1998-09-17 Abbott Laboratories Gene humain de metalloprotease matricielle, proteines codees a partir de ce dernier, et procedes d'utilisation associes
US6399371B1 (en) 1997-03-11 2002-06-04 Abbott Laboratories Human matrix metalloprotease gene, proteins encoded therefrom and methods of using same

Also Published As

Publication number Publication date
WO1997019178A3 (fr) 1997-09-25
DE19543265A1 (de) 1997-06-05

Similar Documents

Publication Publication Date Title
DE69831754T2 (de) Zelloberflächenglycoproteinen zusammenhang mit menschlicher b zell lymphomen - ulbp, dns und polypeptiden
DE69926480T2 (de) Acpl dna und polypeptide
Sur et al. Localization of eosinophil‐derived neurotoxin and eosinophil cationic protein in neutrophilic leukocytes
EP1003781B1 (fr) Proteines de fixation de l'interleukine-18, leur preparation et utilisation
DE69117949T2 (de) Mit akuter pankreatitis assoziiertes protein, mittel zur diagnose von akuter pankreatitis
DE69229252T2 (de) DNS, welche das Interleukin-1B-Vorläufer-Converting-Enzym kodiert
EP1076710B1 (fr) Facteurs affectant l'activite de l'enzyme liberant le recepteur du facteur de necrose tumorale
JP2003527067A (ja) Ａｃｒｐ３０（３０ｋｄの脂肪細胞補体関連タンパク質）の相同体ａｃｒｐ３０ｒ１ｌ
EP1381628B1 (fr) Peptides specifiques d'inflammations et leurs utilisations
JP3996191B2 (ja) Ｃｃ型ケモカイン様タンパク質
EP0936270A2 (fr) Sérine protéase de la prostate
EP2149051B1 (fr) Procédé et agents d'immunoabsorption permettant la détection spécifique et l'absorption d'anticorps associés à la maladie coeliaque et à la dermatite herpétiforme
US20030092614A1 (en) ADPI-41, a novel protein isolated from brain tissue homogenate and uses therefor
Popken-Harris et al. Biochemical properties, activities, and presence in biologic fluids of eosinophil granule major basic protein
WO1997019178A2 (fr) Proteases metalliques autoantigenes et procedes permettant de diagnostiquer des maladies auto-immunes
Plager et al. A novel human homolog of eosinophil major basic protein
WO1998017800A1 (fr) Larc, nouvelle chimiokine cc humaine
JPH11243960A (ja) ヒトｃｃ型ケモカインエオタキシン３
JP2003526322A (ja) 分泌型システインリッチタンパク質−６（ｓｃｒｐ−６）
JP2005502337A (ja) 融合タンパク質
EP3149485B1 (fr) Procédé de diagnostic d'une maladie médiée par la voie alterne du système du complément ou d'un risque pour celle-ci
EP0682113A2 (fr) Polypeptide produit dans une lignée cellulaire endothéliale et ADN codant pour celle-ci
JPH1142093A (ja) シアロアドヘシンファミリーメンバー−３
JP2002501744A (ja) Ｗｎｔ−１０ａ
DE60122721T2 (de) Diagnostische verwendungen des t-zell proteins tzon7, der davon abgeleiteten peptide und des antikörpers

Legal Events

Date Code Title Description
AK Designated states

Kind code of ref document: A2

Designated state(s): CA GB JP US

AL Designated countries for regional patents

Kind code of ref document: A2

Designated state(s): AT BE CH DE DK ES FI FR GB GR IE IT LU MC NL PT SE

DFPE Request for preliminary examination filed prior to expiration of 19th month from priority date (pct application filed before 20040101)
121 Ep: the epo has been informed by wipo that ep was designated in this application
NENP Non-entry into the national phase

Ref country code: JP

Ref document number: 97519273

Format of ref document f/p: F

122 Ep: pct application non-entry in european phase
点击 这是indexloc提供的php浏览器服务，不要输入任何密码和下载